In 2009, at Oregon National Primate Research Center in Beaverton,
Oregon, Shoukhrat Mitalipov, Masahito Tachibana, and their team of
researchers replaced the mitochondrial genes of primate embryonic stem
cells via spindle transfer. Spindle replacement, also called spindle
transfer, is the process of removing the genetic material found in the
nucleus of one egg cell, or oocyte, and placing it in another egg that
had its nucleus removed. Mitochondria are organelles found in all cells
and contain some of the cell’s genetic material. Mutations in the
mitochondrial DNA can lead to neurodegenerative and muscle diseases.
Mitalipov and Tachibana used spindle replacement to produce healthy
offspring from an egg with mutated mitochondria in rhesus macaques
(Macaca mulatta). The experiment showed that spindle transfer eliminated
the chance of transmission of mitochondrial diseases from the affected
primates to their offspring, offering the potential to eliminate
mitochondrial diseases in humans.

The experiment focused on
mitochondria, which are energy-producing organelles found in all
eukaryotic cells that are important to normal development. The
mitochondria contain thirty-seven genes responsible for energy
production in cells. Occasionally, the genetic material found in the
mitochondria contains mutations. Mitochondria are maternally inherited,
meaning they are passed from mother to offspring. Mitochondria contain
approximately .01 percent of a cell’s genetic material, which is called
mitochondrial DNA. Each mitochondrion contains between two and ten
copies of mitochondrial DNA. Each cell, containing many mitochondria,
can contain thousands of copies of mitochondrial DNA. Because of the
large number of copies of mitochondrial DNA, mutations occur in the
mitochondrial DNA ten times more often than in the nuclear genome, the
DNA of the cell’s nucleus. Researchers associate many mutations of
mitochondrial DNA with specific disorders, including myopathies, or
muscular disorders, neurodegenerative diseases, and diabetes.

To
identify mitochondrial DNA mutations within a cell, researchers analyze
the genetic material of mitochondria from that cell. A cell normally
contains many copies of the same mitochondrial DNA, called homoplasmy.
When a cell contains two or more types of mitochondrial DNA, called
heteroplasmy, researchers are alerted that mutated mitochondria are
present. When enough cells contain mitochondrial mutations, different
tissues can be affected, which can result in mitochondrial diseases.

In
2009, Mitalipov and Tachibana studied rhesus macaque eggs with
mitochondrial DNA mutations. The researchers used the technique of
spindle transfer to successfully transfer the nuclear DNA in the
affected egg into a donor egg containing healthy mitochondrial DNA.
Mitalipov and Tachibana hypothesized that nuclear DNA from an egg with
mutated mitochondrial DNA could be transplanted into an egg with healthy
mitochondrial DNA through the use of spindle transfer technique, which
would eliminate the possibility of passing the mutated mitochondrial DNA
from mother to offspring. When using spindle transfer, researchers first
remove the genetic material from an egg cell that contains
heteroplasmic, or a mix of healthy and mutated mitochondrial DNA. Then
the researchers transplant that egg cell’s genetic material into a donor
egg cell whose nucleus has been removed and whose mitochondrial DNA is
undamaged. Following the transfer of genetic information to the egg and
after a sperm has fertilized the egg cell, the resulting embryo will
develop with healthy mitochondria.

To begin, Mitalipov and Tachibana
investigated the distribution of mitochondria around the cell’s
cytoplasm within mature rhesus monkey eggs to prevent transferring over
mutated mitochondria. They found that mitochondria were evenly
distributed across the cytoplasm. They also noted that chromosomes,
which contain nuclear DNA, and spindle fibers that separate the
chromosomes, lacked mitochondria in the cytoplasm that surrounds them.
Those observations led Tachibana and Mitalipov to predict that they
could transfer the spindle and chromosomes of a cell without also
transferring the cytoplasm containing the damaged mitochondrial DNA.
That would enable them to avoid transferring mutated mitochondria into
the donor egg. To test their prediction they removed the
spindle-chromosomal complex surrounded by a small amount of cytoplasm, a
complex called the karyoplast. Tachibana and Mitalipov calculated that
only 1.5 percent of the cell’s cytoplasm was included in the karyoplast
samples, a percentage they determined to be negligible. The researchers
concluded that the small amount of mutated mitochondria contained in the
1.5 percent was not enough to affect the outcome of the egg cell.

Next,
Mitalipov and Tachibana investigated the process of transplanting the
karyoplast into donor cells with their nucleus removed. The team
inserted the karyoplast into the egg cell. After placement of the
karyoplast in the egg cell, the investigators attempted to facilitate
the fusion of the karyoplast to the cell’s cytoplasm. To do that, they
applied electrical stimulation to the egg cell to encourage
permeability, the ability of objects to pass through the cell’s membrane.
After the fusion of the karyoplast with the donor cytoplasm, Mitalipov
and Tachibana observed a problem when the egg cell began meiosis, the
process of cell division of reproductive cells that should only occur
after the egg is fertilized. The researchers observed premature meiosis.
They found that the unintended division of the egg cell produced a polar
body, which is a cell that cannot develop into a mature egg.

Mitalipov and Tachibana hypothesized that the electrical stimulation
they used to fuse the karyoplast with the donor cell caused premature
meiosis, and that they could avoid premature meiosis by using
SeV-assisted fusion. SeV-assisted fusion is an alternative method of
karyoplast fusion that involves the use of an extract from the Sendai
virus, or SeV. The researchers briefly exposed the karyoplast to the
viral extract. They found that the viral extract enabled rapid fusion of
the karyoplast and the surrounding cytoplasm of the donor egg. The
resulting egg successfully remained in its mature state and did not
undergo premature meiosis. The researchers concluded that SeV-assisted
fusion prevented the premature division of the egg and was therefore a
valid means of karyoplast fusion.

Mitalipov and Tachibana then compared
the development competence, or the ability of the egg to develop
further, of the two processes of karyoplast fusion. They fertilized eggs
that underwent electroporation-assisted fusion and eggs that underwent
SeV-assisted fusion. The fertilized electroporation-assisted eggs failed
to create a pronucleus, the combined nucleus of the sperm and egg cells.
Without a pronucleus, the egg would not form and embryo. The fertilized
SeV-assisted eggs underwent successful fertilization and development.
Those results confirmed that SeV-assisted fusion did not compromise
fertilization and embryonic development. That outcome showed that the
technology was successful in producing healthy, viable blastocysts. A
blastocyst is a clump of cells in the early stage of development in
mammals. They contain an outer ring of cells and an inner cell mass.

To
assess the quality of the blastocysts produced by SeV-assisted fusion
and fertilization, Mitalipov and Tachibana compared the spindle transfer
blastocysts to a control group of unaltered blastocysts. The researchers
found that the spindle transfer blastocysts contained similar numbers of
inner cell mass cells and total number of cells compared to the
unaltered blastocyst control group. The comparison showed that the
blastocysts developed using their technology were as healthy as
blastocysts that did not undergo mitochondrial transfer.

To further
examine the development potential, Mitalipov and Tachibana isolated
embryonic stem cell lines, or cells with the potential to develop into
many cell types. The team isolated them from the spindle transfer
blastocysts, as well as from the control group to ensure that the stem
cell lines developed into healthy cell lines containing many cell types.
Both cell lines exhibited similar levels of pluripotency, or the ability
for one type of cell to become many different cell types. The ability of
embryonic cells to develop into many cell types is crucial in the
development of an embryo.

The researchers then checked to see whether
spindle transfer caused chromosome abnormality. Chromosome abnormality
occurs when a cell has an abnormal number of chromosomes or when
chromosomes are malformed. Observing chromosome abnormality would
indicate that the technique damaged chromosomes. The analysis demonstrated
that the spindle transfer chromosomes had no abnormalities when
compared to the control group.

Next, Mitalipov and Tachibana tested the
developmental potential of the spindle transfer blastocysts to see
whether they would result in embryos that could be implanted in a female
rhesus macaque. They fertilized the spindle transfer blastocysts and
transplanted the resulting embryos into the reproductive tracts of two
recipient female macaques. On 24 April 2009, the first female macaque, which
Tachibana and Mitalipov had implanted with the fertilized egg, gave birth to a set of healthy twin
macaques. According to Tachibana, the twins were the first spindle
transfer animals to be born with healthy mitochondrial DNA. On 8 May
2009, the second pregnant macaque gave birth to a healthy infant. The
three infants were all of healthy birth weight and size. Mitalipov and
Tachibana concluded that karyoplasts could be successfully isolated and
transplanted into healthy, enucleated oocytes.

Finally, the team sought
to determine if any mutated mitochondrial DNA had managed to form in the
mitochondrial DNA of the resulting offspring. Mitalipov, Tachibana, and
their team found that the nuclear DNA of the offspring matched the
genome of the spindle-donor animals. Further, they confirmed that
damaged mitochondrial DNA was not present in the mitochondria of the
offspring.

Overall, the results of the experiment showed that scientists
could replace mutated mitochondrial DNA in mature eggs using the spindle
transfer technique. Mitalipov states that the technology has the
potential to eliminate the chances of a woman affected by a
mitochondrial disease passing the mutation on to her offspring. Although
the experiment documented the first case of spindle transfer in rhesus
macaques, Mitalipov argues that one day the technology could be applied
to humans. The technology can serve as a new therapeutic approach to
eliminating the mitochondrial disease before the offspring are born.